DESCRIPTION: (Applicant's Abstract) The broad, long-term objective of this R21 application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by a putative endocannabinoid uptake system. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process would play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aims are (1) To isolate and identify the protein(s) responsible for the uptake of endogenous cannabinoids, and (2) To characterize the transport activity associated with anandamide uptake. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including psychosis, motor movement, inflammation, blood pressure, and pain. Based upon data indicating functional similarities between anandamide transport and the uptake of long-chain fatty acids, the Research Design is to explore the potential contributions of various fatty acid transporters in endocannabinoid transport using heterologous expression systems. In addition, the pharmacologic profile of endocannabinoid uptake in native and transfected cells will be determined, and the tissue distribution of the identified anandamide transporter investigated to better understand the role of transport proteins in endocannabinoid signaling. The Methods to be used include molecular cloning techniques, characterization of transport proteins using radiolabeled substrate uptake assays, determinations of intracellular accumulation of endocannabinoid substrates using mass spectrometry, and in situ hybridization to reveal transporter distribution. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.